1-Butanol, as a next-generation biofuel, is an important target product of biorefinery research. With the introduction of the CoA-dependent pathway or Ehrlich pathway, many engineered strains have been developed to produce 1-butanol, although cofactor imbalance occurred in engineered strains by the introduction of the 1-butanol synthesis pathway. Several studies have been performed to regenerate NADH by overexpressing NAD(+)-dependent enzymes. However, the significant role of cofactor regeneration in 1-butanol production and the transcription level of the target genes have seldom been studied. The 1-butanol producer, recombinant Klebsiella pneumoniae (KLA), was constructed by overexpressing the genes kivd, leuABCD, and adhE1 under the control of tac promoter in this study, and several NADH regeneration strategies were adopted to solve the problem of NADH imbalance, including the introduction of NAD(+)-dependent enzymes (formate dehydrogenase, pyridine nucleotide transhydrogenase, and glucose dehydrogenase) or elimination of the NADH competition pathway (1,3-propanediol synthesis). The resultant NADH/NAD(+) ratio, 1-butanol production, and transcription levels have been significantly affected. In comparison to the wild-type strain, the NADH/NAD(+) ratio in the reengineered strains was increased by 78-135%, and the transcript levels of target genes have been obviously interfered. Moreover, the resultant 1-butanol titer was increased by 83-114% in comparison to KLA.